Daniel:Notebook/ComboLock/2016-8-29
Positive Control Dilution Test (Started Friday)
Protocol
Today I'm testing the samples using the P2 and P12RC amplification primers. Yesterday included the original amplicon materials; this qPCR should be only the DNA still attached to beads.
- qPCR X2-Primers
- Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
- Load lanes according to table
- Master Mix
- 352.5 uL KAPA SyberFast MM
- 310.2 uL nfH2O
- 7.05 uL primer2
- 7.05 uL primer12RC
- Add 48 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x32
- 72C 2 min
- 16C hold
- TBE Gel
- Mix together 72 uL TBE and 18 uL 6X loading dye in a 0.2mL tube
- Add 1 or 2 uL sample according to table
- Add 10 uL mix to well
- Run gel for 25 minutes at 250V
- Stain for 3 min with 3 uL SYBR gold
- Image in gel doc
Sample Name | Sample No. | qPCR Lane |
Pure C1-C2 | Sample 00 | A1 |
1E+11 | Sample 0 | B1 |
1E+10 | Sample 1 | C1 |
1E+09 | Sample 2 | D1 |
1E+08 | Sample 3 | E1 |
1E+07 | Sample 4 | F1 |
1E+06 | Sample 5 | G1 |
1E+05 | Sample 6 | H1 |
10000 | Sample 7 | A12 |
1000 | Sample 8 | B12 |
100 | Sample 9 | C12 |
10 | Sample 10 | D12 |
1 | Sample 11 | E12 |
NTC | NTC | F12 |
Gel Lane | Sample Name | qPCR Lane | uL Sample |
2 | 25 bp Ladder | 2 | |
3 | 1E11 Molecule | B1 | 1 |
4 | 1E9 Molecule | D1 | 1 |
5 | 1E7Molecule | F1 | 2 |
6 | 1E5 Molecule | H1 | 2 |
7 | 1000 Molecule | B12 | 2 |
8 | 10 Molecule | D12 | 2 |
9 | 1 Molecule | E12 | 2 |
Results
- 20160829-X2Dilution-Primers-qPCR.png
qPCR Curves
- 2016-08-29-X2Dilution-PrimerAmpGel.png
Representative Gel Image
X3 Dilution Test
Based on the results from the X2 dilution test, I have a couple of factors I'd like to change for testing X3. From the gel results yesterday, I'd say that I couldn't see signal below 10^9 molecules, which isn't great. However, I'm guessing that this is probably due to loss of bead signal (I lost too many beads). So, I'm going to do 2 things: (1) double the number of beads. This won't actually do anything except make it easier for me to see the beads. The number of beads shouldn't change the results, only the number of targets. (2) Technical replicates. Sometimes n=1 isn't a great idea. So I'll do n=3. And finally, (3rd bonus point!) I'm going to do fewer samples and only test in the higher numbers (0-5 which ranges from 1E11 to 1E6 molecules, respectively).
Buffers
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 3 |
dNTPs | 100 uM | 3 |
Hemo Klentaq | NA | 6 |
Amp Ligase | 5 U/uL | 3 |
nfH2O | NA | 15 |
Protocol
Tube No (A-C) | Dilution | Molecules Available |
0 | 1 | 100000000000 |
1 | 10 | 10000000000 |
2 | 100 | 1.E+09 |
3 | 1000 | 1.E+08 |
4 | 10000 | 1.E+07 |
5 | 100000 | 1.E+06 |
- Latch and Padlock Hybridization
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
- Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with intermittent mixing
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 30 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow